Project Summary Through substrate oxidation, mitochondria generate a high potential across the inner mitochondrial membrane (IMM). The energy of this potential is usually converted into ATP by the mitochondrial ATP synthase, but a fraction is dissipated as heat due to the presence of a H+ leak across the IMM. This mitochondrial H+ leak is mediated by specialized proteins of the IMM, such as uncoupling protein 1 (UCP1), and it has important physiological functions. It controls the metabolic efficiency of the body, helps to support the core body temperature, and reduces mitochondrial production of reactive oxygen species to protect against oxidative damage. The mitochondrial H+ leak is considered to be important in protective mechanisms against obesity, diabetes, and age-related degenerative disorders as well as against pathological conditions involving mitochondrial oxidative stress such as ischemia-reperfusion. Despite its physiological and pathophysiological significance, the mitochondrial H+ leak remains poorly understood, primarily due to the lack of direct methods to study it. We recently developed a method that removes this technical barrier and for the first time allows direct patch-clamp recording of H+ leak currents from the whole IMM. This method helped us resolve long- standing questions about the mechanism of the UCP1-dependent thermogenic H+ leak across the IMM of brown fat. In this application, we propose to use the patch-clamp technique to further characterize the mitochondrial H+ leak in several tissues that play important roles in thermogenesis and energy metabolism. The specific aims of this proposal are to: 1) identify the protein(s) responsible for the mitochondrial H+ leak in non-adipose tissues; 2) characterize the mechanism of the fatty acid-activated mitochondrial H+ leak via the adenine nucleotide translocator (ANT); 3) characterize the mechanism of the mitochondrial H+ leak induced by protonophores DNP and FCCP. Accomplishment of these specific aims will help us elucidate the principal mechanism that regulates metabolic efficiency and thermogenesis, and such knowledge will aid the development of therapeutic interventions to control obesity, diabetes, and age-related degenerative disorders.